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Inhibitor of differentiation (Id) is a member of the helix-loop-helix protein family and exerts negative transcriptional regulation by forming heterodimers with other HLH proteins to inhibit gene expression. Id can promote cell proliferation and inhibit cell differentiation. Especially, Id plays an essential role in the development and differentiation of various immune cells. This review mainly introduces the latest research progress in how members of the Id family regulate the generation and fate determination of multiple cell lineages in the innate and adaptive immune systems. By interacting with different transcription factors such as E protein, Id has a variety of functions in the differentiation process of natural killer cells, innate lymphoid cells, T cells, and B cells, etc., ensuring the well-coordinated development of immune cells in various immune organs. On the other hand, the abnormal expression and functional deficiency of the Id gene are closely related to the occurrence and progression of hematologic malignancies. In different hematologic malignancies ranging from leukemia to malignant lymphoma, Id plays diverse roles as a cancer-promoting or tumor suppressor factor. Therefore, Id can be used as a marker for the diagnosis and prognosis of hematologic malignancies and has the potential to become an important target for cancer therapy.
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Objective • To study the effect of inhibitor of differentiation 1 (ID1) on ocular neovascularization. Methods • The oxygen-induced retinal neovascularization (OIR), laser-induced choroidal neovascularization (CNV) and over-expression of vascular endothelial growth factor (VEGF) (Rho-VEGF) transgenic mice were established. The localization and mRNA level of ID1 in retina of OIR mice and Rho-VEGF transgenic mice were determined by immunofluorescence staining and quantitative real-time PCR. Mice deficient in ID1 (ID1-/-) were used to induce retinal neovascularization in accordance with the above three models, and to compare the changes of ID1 on the number of retinal, subretinal and choroidal neovascularization areas. In order to explore the role ID1 in neovascularization, the numbers and areas of retinal, subretinal and choroidal neovascularization in the mice models with or without ID1 deficiency were compared. Its effect on the related factors, i.e. hypoxia-inducible factor-1α (HIF-1α), VEGF and vascular endothelial growth factor receptor 1/2 (VEGFR1/2) were also observed. Results • Mice deficient in ID1 showed a significant reduction in the area of neovascularization in these three models(P<0.05). Mice lacking ID1 showed reduced levels of HIF-1α, VEGF and VEGFR 1. Conclusion • ID1 promotes the expression of HIF-1α, VEGF and VEGFR1 in the retina and choroidal neovascularization during hypoxia and oxidative injury.
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Objective · To study the effect of inhibitor of differentiation 1 (ID1) on ocular neovascularization. Methods · The oxygen-induced retinal neovascularization (OIR), laser-induced choroidal neovascularization (CNV) and over-expression of vascular endothelial growth factor (VEGF) (Rho-VEGF) transgenic mice were established. The localization and mRNA level of ID1 in retina of OIR mice and Rho-VEGF transgenic mice were determined by immunofluorescence staining and quantitative real-time PCR. Mice deficient in ID1 (ID1-/-) were used to induce retinal neovascularization in accordance with the above three models, and to compare the changes of ID1 on the number of retinal, subretinal and choroidal neovascularization areas. In order to explore the role ID1 in neovascularization, the numbers and areas of retinal, subretinal and choroidal neovascularization in the mice models with or without ID1 deficiency were compared. Its effect on the related factors, i.e. hypoxia-inducible factor-1α (HIF-1α), VEGF and vascular endothelial growth factor receptor 1/2 (VEGFR1/2) were also observed. Results · Mice deficient in ID1 showed a significant reduction in the area of neovascularization in these three models (P＜0.05). Mice lacking ID1 showed reduced levels of HIF-1α, VEGF and VEGFR 1. Conclusion · ID1 promotes the expression of HIF-1α, VEGF and VEGFR1 in the retina and choroidal neovascularization during hypoxia and oxidative injury.
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@# Objective: To investigate whether inhibitor of differentiation 1 gene (Id1) and Id3 gene can synergistically promote epithelial-mesenchymal transition (EMT), invasion and migration of colon cancer SW620 cells and to explore its underlying mechanisms. Methods: The SW620 cell strain with Idl or Id3 gene knockdown and the SW620 cell strain with Id1/Id3 gene double-knockdown were constructed by lentiviral vectors transfection. The SW620 cells were divided into four groups, which included SW620-Sh-Id1 group (transfected with shRNA-Id1), SW620-Sh-Id3 group (transfected with shRNA-Id3), SW620-Sh-Id1-Id3 group (transfected with shRNAId1 plus shRNA-Id3) and SW620-NC group (transfected with negative lentivirus). The efficiency of knockdown was detected by Realtime qPCR and Western blotting. The influence of stable knockdown of Idl or Id3 on cell morphological change was observed under a microscope. The changes of migration and invasion abilities of the SW620 cells were determined by wound healing assay and Transwell assay. EMT, invasion and migration related proteins were measured by Western blotting. Results: The SW620 cell strains with Idl and/or Id3 gene knockdown were successfully constructed. Idl and Id3 knockdown induced the epithelial-like to the mesenchymanl-like transformation of SW620 cells. (1) Compared with the control group, the invasion and migration abilities of the SW620 cells were significantly decreased in the SW620-Sh-Id1 group and SW620-Sh-Id3 group (all P<0.05). (2) Meanwhile, the invasion and migration abilities in the SW620-Sh-Id1-Id3 group were obviously weaker than the SW620-Sh-Id1 group and SW620-Sh-Id3 group (all P<0.05). (3) Compared with the control group, the SW620-Sh-Id1 group and SW620-Sh-Id3 group had a reduction in the protein expressions of βcatenin, snail1 and MMP2, and an increase in the protein expressions of E-cadherin and TIMP2 (all P<0.05). (4) Compared with the SW620-Sh-Id1 group and SW620-Sh-Id3 group , the protein expressions of β-catenin, snail1 and MMP2 were reduced, and the protein expressions of E-cadherin and TIMP2 were increased in the SW620-Sh-Id1-Id3 group (all P<0.05). Conclusion: Id1 and Id3 could synergistically influence invasion and migration of SW620 cells, possibly through inducing EMT.
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Objective To investigate the protective effect of mild hypothermia on cerebral ischemia-reperfusion injury in rats and the effect of mild hypothermia on the expression of inhibitor of differentiation 2 (Id2) protein.Methods A total of 72 adult male rats were randomly divided into a sham operation group,a normothermia group,and a mild hypothermia group.A model of middle cerebral artery occlusion was induced by a suture method.The mild hypothermia group was treated with low temperature (anal temperature 33±1 ℃,tympanic membrane temperature 31±1 ℃).Modified Neurological Severity Score (mNSS) was used to evaluate neurological deficits,triphenyltetrazolium chloride staining was used to detect infarct volume,and Western blot was used to detect the Id2 expression in the ischemic cortex at ischemia-reperfusion 6,12,24,and 72 h,respectively.ResultsThe mNSS scores in the mild hypothermia group were significantly lower than those in the normothermia group,the infarct volumes were significantly smaller than those in the normothermia group at ischemia-reperfusion 6,12,24,and 72 h (all P<0.001).Western blot analysis showed that the Id2 expressions in the ischemic cortex in the mild hypothermia group were significantly lower than those in the normothermia group at ischemia-reperfusion 6,12,24,and 72 h (all P<0.05).Conclusion s Mild hypothermia can decrease neurological deficits and reduce infarct volume after cerebral ischemia-reperfusion,its mechanism may be associated with the down-regulation of the Id2 expression.
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Objective: To investigate the effect of inhibitor of differentiation 1 (Id 1) gene on apoptosis of human osteosarcoma MG63 cells, and to explore its possible molecular mechanism. Methods: The specific siRNA targeting Id 1 gene (Id1-siRNA) was infected into MG63 cells using recombinant adenovirus Ad-siRNA-Id1 as the AdsiId1 group, while the negative control group (infection with the empty vector adenovirus AdRFP) and the blank control group (non-infection) were set up, respectively. The expression levels of Id1 mRNA and protein were detected by semi-quantitative RT-PCR and Western blotting, respectively. The apoptosis of MG63 cells was detected by Annexin V-FITC staining and DAPI staining. The cell cycle distribution of MG63 cells was detected by FCM method. The expression levels of apoptosis-related proteins (Bcl-2 and survivin) and Wnt pathway-related proteins [β-catenin, receptor tyrosine kinase-like orphan receptor 2 (ROR2) and CCAAT/enhancer-binding protein homologous protein (CHOP)] were detected by Western blotting. Results: After infection with recombinant adenovirus Ad-siRNA-Id1, the expression levels of Id1 mRNA and protein in MG63 cells were significantly decreased (both P < 0.05). The apoptotic rate in AdsiId1 group was higher than those in AdRFP and the blank control groups (both P < 0.05). The expression levels of Bcl-2 and survivin mRNAs and proteins were significantly decreased (all P < 0.05). The expressions of β-catenin, ROR2 and CHOP proteins were also significantly decreased (all P < 0.05). Conclusion: The silencing of Id 1 gene expression can promote the apoptosis of osteosarcoma cells, which may be related to regulating Wnt signaling pathway.
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Objective The inhibitor of differentiation 3 (Id3) is an important transcriptional regulation factor, which participates in tumorigenesis, cell proliferation, and cell apoptosis.β-catenin, as a central molecule of the Wnt signaling pathway, is critical for tumor development.This study aimed to evaluate the expressions of these two molecules and the regulatory effect of Id3 on β-catenin in different tumor cells.Methods Total RNA was extracted using the Trizol Reagent.The relative mRNA expression levels of Id3 and β-catenin in tumor cells were detected by quantitative real-timePCR(qRT-PCR).The recombinant eukaryotic expression vector pEGFP/Id3 with the human Id3 gene was transfected into A549, A549/ DDP and SW-480 cells using the non-liposome-mediated method.The protein expressions of Id3 and β-catenin were determined by Western blot.Results The expression of Id3 was significantly lower in the colorectal cancer cell lines SW-480 and HT-29 than in A549 and other tumor cells (P0.05).Western blot showed the same results.Conclusion The expression levels of Id3 and β-catenin vary in different tumor cell lines.Anabnormally high level of β-catenin is an important risk factor for colorectal cancer, and the down-regulatedexpression of β-catenin after eogenous transfection of Id3 may provide some new ideas for target gene therapies of colorectal cancer.
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AIM: To investigate the effects of bone morphogenetic protein 7 ( BMP-7 ) on the expression of transcription factor E2A and inhibitor of differentiation 2 (Id2) in the renal tubule epithelial cells(NRK-52E)exposed to high glucose, and to explore its possible mechanism of improving renal tubular fibrosis induced by high glucose.METH-ODS:The NRK-52E cells were divided into control group, high glucose (HG) group and high glucose with different doses of BMP-7 (10μg/L and 20μg/L) group.The cells in HG group and BMP-7 group were cultured for 12 h, 24 h and 48 h. The protein expression of Id2, E2A, E-cadherin,α-smooth muscle actin (α-SMA) and collagen-I was detected by Western blot.In addition, the mRNA expression of Id2 was detected by real-time PCR.RESULTS:Compared with control group, the mRNA and protein levels of Id2 and the protein level of E-cadherin were down-regulated, while the protein levels of E2A,α-SMA and collagen-I were up-regulated in HG group (P<0.05).Compared with HG group, the mRNA and pro-tein levels of Id2 and the protein level of E-cadherin were significantly up-regulated, while the protein expression of E2A,α-SMA and collagen-I was significantly down-regulated in 20 μg/L BMP-7 group ( P<0.05 ) .The correlation analysis showed that the Id2 protein level was negatively correlated with the E2A protein level (P<0.05).CONCLUSION:BMP-7 may intercept the process of renal tubule fibrosis induced by high glucose via promoting the expression of Id2 and inhibi-ting the expression of E2A at protein level.
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Objective: To explore the role and the possible mechanism of inhibitor of differentiation 1 (Id1) in angiogenesis of colon cancer. Methods: After transfection with Id1 over-expression vector plasmid, the expression levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) mRNAs and proteins in HT-29 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The ability of tube formation of human umbilical vein endothelial cells (HUVECs) after culture with supernatant of HT-29 cells with Id1 overexpression for 24 h was observed by tube formation experiment. The tumorigenesis of HT-29 cells with Id1 over-expression in nude mice was observed, and the microvessel density (MVD) of transplanted tumor tissues was detected by immunohistochemistry, . Results: The expression levels of HIF-1α and VEGF mRNAs and proteins in HT-29 cells with Id1 over-expression were higher than those in the HT-29 cells transfected with empty vector (negative control) and the HT-29 cells without any transfection (blank control) (all P < 0.05). The number of tube formation of HUVECs after culture with supernatant of HT-29 cells with Id1 over-expression was higher than those of the negative control and the blank control groups (both P < 0.05). The ability of tmorigenesis and the number of MVD in Id1 overexpression group were both higher than those in the negative control and the blank control groups (all P < 0.05). Conclusion: Id1 may promote the angiogenesis of colon cancer by regulating the expressions of HIF-1α and VEGF.
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Purpose To study the expression of inhibitor of differentiation 2 (Id2) in Ewing sarcoma of bone as well as the correlation with cell cycle proteins. Methods The expression of Id2, c-Myc, Cyclin D1, p53, p16 and p27 in 45 cases of Ewing sarcoma were detected by immunohistochemical EnVision methods, the relationship between the expression of Id2 and clinicopathologic characteristics and the correlation with cell cycle proteins were analyzed. Results Total expression of Id2 was found in 88. 9% of Ewing sarcoma. Strong expression of Id2 was detected in 33. 3% cases. Higher frequencies of c-Myc and Cyclin D1 immunostaining were observed (66. 7% and 71. 1%, respectively), but lower frequencies of p53, p16 and p27 expression were present (20. 0%, 35. 6% and 28. 9%, respectively). Id2 overexpression displayed a negative relationship with p27 expression (P<0. 05). Among the follow-up of 24 cases, overexpression of Id2 was found more frequency in dead and metastases cases (41. 2%) when compared to that of progres-sion-free survival cases (14. 3%). Conclusions Id2 was extensively expressed in Ewing sarcoma of bone. Overexpression of Id2 and its correlation with p27 expression suggest that this is an important pathway involved in development of Ewing sarcoma. Moreover, Id2 overexpression may predict poor prognosis.
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Objective To investigate the expressions and clinical significance of Id1 in diffuse large B‐cell Lymphomas (DL‐BCL) tissue and Id1 gene in bone warrow cell .Methods Forty cases of DLBCL(observation group) and 25 cases of reactive lymph‐oid hyperplasia (control group) were included in this study which were admitted by our hospital from October 2011 to October 2014 .The expression of Id1 proteins in DLBCL and reactive lymphoid hyperplasia were detected by imunohistochemical technique . The expression of Id1 genes in all patients′marrow cells was detected by reverse‐transcriptase polymerase chain reaction .The data were collected and analyzed by designed person .Results In 40 DLBCLs ,the positive rate of Id1 were 75 .00% (30/40) ,which was higher than in RHs 32 .00% (8/25) ,with statistic difference(P= 0 .001) .Id1 protein was not correlated with sex and age(P>0 .05) ,but was correlated with clinical stage ,LDH level and extranodal infiltration(P<0 .05) .The expression of Id1 genes in mar‐row cells in DLBCL was higher than in RH (Id1mRNA level 2 .80 ± 0 .87 vs .1 .37 ± 0 .51 ,P<0 .05) ,and also correlated with clini‐cal stage ,LDH level and extranodal infiltration(P<0 .05) .Conclusion The expression of Id1 proteins and genes are much higher in DLBCL tissue and marrow and probably related to the prognosis of DLBCL .This discovery would contribute to predicting prognosis of DLBCL ,w hich also could be a therapeutic target of DLBCL in the future .
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Objective To observe the expression level of inhibitor of differentiation 2 (Id‐2) and matrix metalloproteinases‐9 (MMP‐9) in rectal cancer ,analysis the correlation of the expression level of them ,to study the relationship between the expression level of them and the clinical pathology indicators of rectal cancer .Methods Rectal cancer tissues and normal tissue adjacent to rec‐tal cancer were obtained from the rectal cancer resection of 56 patients with rectal cancer ,using immunohistochemical method to ob‐serve the expression level of Id‐2 and MMP‐9 in normal tissue adjacent to rectal cancer and rectal cancer and Spearman correlation test to detect the correlation between the expression level of Id‐2 and MMP‐9 ;then we analyzed the relationships between the ex‐pression level of Id‐2 and MMP‐9 and the index of rectal cancer clinical pathology .Results The positive expression rate of Id‐2 in the in rectal cancer tissues is more higher than that of normal tissue of adjacent to rectal cancer (73 .21% vs .48 .21% ,P0 .05) .Conclusion There is a close relationship between the expression levels of Id‐2 and MMP‐9 in rectal cancer and the occurrence and development of rectal cancer .Rectal cancer with the higher Id‐2 expression level may be the ways to achieve tumor invasion and metastasis through MMP‐9 as a facilitator .
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Objective To construct short hairpin RNA (shRNA) expression plasmids against the inhibitor of differentiation 2 (Id2) gene and establish a suitable cell model for the role of Id2 in proliferation and differentiation of human Ewing sarcoma cell.Methods Three shRNA sequences targeting Id2 gene were designed and inserted into the pGPU6/GFP/Neo (-shRNA-Id2) expression vectors.The recombinant pGPU6/GFP/Neo-shRNA-Id2 plasmids were introduced into Ewing sarcoma RD-ES cells by liposome-mediated transfection.The knock-down efficiency of Id2 in infected RD-ES cells was verified by Western blot assay.The cell growth and cell cycle changes were evaluated by cell counting and flow cytometry between transfected cells and control cells.Results The Id2 expression decreased 54 ％ and 57 ％,respectively,in RD-ES cell line which were transfected with the shRNA-Id2-543 and shRNA-Id2-593 plasmids compared with the control group cells by Western blot analysis.The cell growth assay demonstrated that the cell number in transfected cells was significantly decreased during 6-7 d compared with the control group (P ＜ 0.05).The cells at the S-phase of cell cycle were increased [(36.60±1.53) ％ and (44.89E2.46) ％ vs (29.73±2.03) ％,P ＜ 0.05],and no significant changes at the G2 phase or even reduction in the transfected cells.Conclusions Id2 stable knock-down cell lines are successfully established.The reduced expression of Id2 is related with decreased cell growth and cell cycle arrest in the S-phase.Id2 maybe plays an important role in proliferation of Ewing sarcoma cell.
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Objective To detect the expression of Id1 and HBx in HBV-related hepatocellular carcinoma (HCC) tissue samples and analyzed the correlation between Id1 expression levels and clinicopathological features of patients.Methods Tumor tissue samples obtained from a total of 113 HCC patients.The expression of Id1 proteins of these samples were detected by immunohistochemical (IHC) staining and evaluated by two independent pathologists.The corrections between the clinical pathological parameters and the IHC scores for Id1and the prognostic significance were statistical analyzed by SPSS 19.0 software.Results Ninty-six of 113 patients is HBV-related HCC.Over-expression of Id1 were found positively correlated with the HBsAg ＞ 200 s/n,histological grade,portal vein invasion.Patients with Id1 overexpression had both shorter disease-free and overall survival times.Conclusions High expression of Id1 was correlated with serum HBsAg,histological grade,portal vein invasion and poor clinical outcomes in HBV-related HCC.
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Objective To construct a lentiviral vector carrying short hairpin RNA (shRNA) of human Id2 gene and to investigate its effect on the proliferation and apoptosis in glioma cell line U251. Methods Four shRNA sequences targeting human Id2 gene were designed and synthesized. The shRNAs were inserted into the pGCSIL-GFP lentiviral vector. Id2 sequence was amplified from the plasmid which contained Id2 gene as verified by PCR, and was inserted into the pEGFP-N1-3FLAG vector. Lentiviral vector for RNAi of Id2 was constructed and was used to infect 293T cells. The infection efficiency was evaluated and then the lentiviral vector was used to infect U251 cellline. Western blotting analysis was employed to assess the gene silencing efficiency. Cell proliferation was tested by MTT. Cell apoptosis was observed by RT-PCR analysis of caspase 3 expression in U251 cells after infection. Results The results of PCR analysis and DNA sequencing agreed to each other. Compared with the control group, the transfected U251 cells had significantly decreased cell proliferation, and increased apoptosis rate and caspase 3 expression (P<0.05).Conclusion We have successfully constructed lentivirus RNAi vector of Id2, which can inhibit the proliferation and promote the apoptosis of glioma cells in vitro.
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Objective The aim of this study is to examine the effect of myoophenolate mofetil (MMF) on epithelial-myofibroblast transiation(EMT) in adenine-induced chronic renal failure (CRF) rat model and the role of vascular endothelial growth factor(VEGF) and inhibitor of differentiation (Id2 and Id3) in EMT in the rat kidney. Methods Sixty-four male Wistar rats were randomly assigned to the following groups: normal control (n=16), CRF (n=24) and MMF(n=24). CRF was induced by gastric gavage of adenine (125 mg·kg-1·d-1) to rats for eight weeks. CRF rats were treated with MMF (15 mg·kg-1·d-1) as "MMF" group. The rats were sacrificed at week 2, 4, 6 and 8, respectively.Urinary protein and serum ereatinine levels were measured, and the histopathologic degrees of interstitial fibrosis were evaluated in Massen-stained sections. Expressions of a-smooth muscle actin (α-SMA),transforming growth factor β1 (TGFβ1), VEGF and Id (Id2 and Id3) in the kidney tissue were assessed by immunohistochemistry, RT-PCR and/or Western blot methods. Results The urinary protein level in MMF group was evidently lower than that in CRF group (P<0.01), whereas no statistically significant difference was observed in serum creatinine level between the two groups. Renal interstitial fibrosis was reduced significantly with MMF treatment (P<0.01). Expression of α-SMA in MMF group was lower than that in CRF rats at week 6, 8 (P<0.01), while expression of TGFβ1 was decreased markedly at week 2, 4,6 (P<0.01). The expressions of VEGF in MMF rats were increased significantly at week 6,8 (P<0.01),and Id2,Id3 in MMF rats were increased significantly at week 4,6 (P<0.05). Conclusions MMF may ameliorate chronic renal fibrosis and EMT in adenine-induced CRF rats. This effect of MMF on EMT is probably related to upregulation of VEGF, Id2 and Id3 expressions and suppressing overexpression of TGFβ1 in renal tissue. The exact mechanism needs to be studied further.
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BACKGROUND: Id proteins are a family of helix-loop-helix proteins and are regarded to be negative regulators of cell differentiation. In general, Id-1 and Id-2 expressions are upregulated during tumor development and progression in a variety of neoplasms, and these expressions may be associated with aggressive tumor behavior. However, little is known about the roles of Id-1 and Id-2 in thyroid neoplasms. METHODS: The expressions of Id-1 and Id-2 were assessed immunohistochemically in 310 normal, hyperplastic, and neoplastic thyroid tissues using tissue microarrays. RESULTS: Normal thyroid tissues rarely expressed Id-1 or Id-2. Moreover, whilst Id-1 expression was more elevated in malignant thyroid tissue than in hyperplastic thyroid tissue, Id-2 expression was more variable. No significant differences were observed between histologic subtypes of thyroid carcinomas with respect to Id-1 or Id-2 expression. Follicular adenomas showed higher expressions of Id-1 and Id-2 than thyroid carcinomas. No significant association was found between clinicopathological parameters and Id-1 expression, though Id-2 expression was significantly reduced in metastatic, stage IV tumors. CONCLUSION: The expressions of Id-1 and Id-2 were elevated in hyperplastic and neoplastic thyroid tissues. However, neither appears suitable as a marker of malignancy or an aggressive phenotype, although Id-2 expression in advanced thyroid carcinomas may reflect a favorable prognosis.
Subject(s)
Humans , Adenocarcinoma, Follicular , Adenoma , Carcinoma, Papillary , Cell Differentiation , Inhibitor of Differentiation Protein 1 , Inhibitor of Differentiation Protein 2 , Phenotype , Prognosis , Thyroid Gland , Thyroid NeoplasmsABSTRACT
Objective:To investigate the inhibitory effect of inhibitor of differentiation 3(Id3)on growth of human lung adenocarcinoma cell line A549.Methods:Recombinant eukaryotic expression vector pEGFP/Id3 was constructed and transfected into A549 cells by liposome-mediated method.Expression of pEGFP/Id3 in A549 cells was analyzed by flow cytometry(FCM),fluorescence microscopy,semi-quantitative RT-PCR and immunocytochemistry.The growth inhibitory rate of A549 cells was examined by MTT assay;cell cycle change was evaluated by PI(propidium iodide)staining method.Cell apoptotic rate and nuclear morphology were detected by Annexin V/7-AAD and Hoechst33258 staining. Results:The recombinant eukaryotic expression vector pEGFP/Id3 was successfully constructed.The expression of EGFP reached the peak 48-72 h after transfection;the expresion of pEGFP-transfected group was higher than that of the pEGFP/ Id3 group.RT-PCR and immunocytochemistry staining showed that Id3 mRNA and protein were effectively expressed in pEGFP/Id3-transfected A549 cells.The growth of cells in pEGFP/Id3 transfeeted cells was significantly inhibited 48-72 h after transfection(P
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Objective: To study mouse Id3(mId3) protein expression in WEHI-231 B lymphoma cells after CD40 ligand(CD40L) treatment. Methods: The mId3 cDNA was amplified from total RNA of WEHI-231 cells by RT-PCR and introduced into the p3FLAG-CMV-10 vector.The 3FLAG-Id3 fusion gene was amplified from p3FLAG-Id3-CMV-10 vector and subcloned into retroviral vector pMX-CITE/IRES-EGFP.Recombinant retrovirus was produced by transient transfection of the Phoenix-ecotropic packaging cell line with pMX-3FLAG-Id3-EGFP vector using FuGene-6 transfection reagent.(WEHI)-231 cells were transduced with recombinant retrovirus.EGFP expression was monitored by flow cytometry.Two cell lines stably expressing 3FLAG-Id3 fusion protein were generated. Results: The proportion of EGFP-positive cells in the population transduced with pMX-3FLAG-EGFP remained constant up to 2 days after transduction,whereas the proportion in the population of pMX-3FLAG-Id3-EGFP-transduced cells decreased with time.After coculturing with NIH3T3 cells or medium alone,the proportion of EGFP-positive cells in pMX-3FLAG-Id3-EGFP-transduced WEHI-231 cells decreased with time,whereas after coculturing with CD40L-NIH3T3 cells,the proportion of EGFP-positive cells in the transduced cells remained constant up to 2 days,suggesting that CD40L could rescue WEHI-231 from Id3 overexpressioninduced growth arrest.After coculturing with CD40L/NIH3T3 or NIH3T3 cells,3FLAG-Id3 fusion protein stably expressing WEHI-231 cells were harvested,lysed,and immunoblotted by using anti-FLAG antibody.The results showed that treatment via CD40 ligation induced significant downregulation of Id3 expression in pMX3FLAG-Id3-EGFP tansduced WEHI 231 cells. Conclusion:CD40 ligation pvevents the Id3 induced cell growth avvest by the dousn-regulation of Id3 expression.
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Objective: To express and purify the inhibitor of differentiation(Id3) in E.coli.Methods: The DNA fragment in the coding region of the human Id3 gene was amplified by PCR and cloned into the pGEM-T Easy vector for sequencing.The Id3 cDNA fragment was then subcloned into the prokaryotic expression vector(pET-32a)(+) and transformed into E.coli BL 21(DE3).The expression of the histidine-tagged(His-Tag) fusion protein was induced with isopropy-?-D-thiogalactoside(IPTG),confirmed by Western blot and purified by the immobilized Ni2+ absorption chromatographic column.Results: The prokaryotic expression vector of Id3 was successfully constructed.Western blot confirmed the expression of the His-Tag fusion protein in E.coli BL 21(DE3) and that of the Id3 fusion protein with the relative molecular mass size of 33 000 after purified by the affinity chromatographic column. Conclusion: Recombinant Id3 can be expressed in E.coli BL 21(DE3) and the obtained fusion protein can be purified by the Ni-affinity chromatography column.